The present invention relates to methods, reagents and kits to sequence nucleic acids or to determine nucleotide changes in nucleic acids. In particular, the present invention relates to using the ribosome together with protein and nucleic acid components of the cellular ribosomal translation system to acquire nucleic acid sequence information.
The primary sequences of nucleic acids are crucial for understanding the function and control of genes and for applying many of the basic techniques of molecular biology. Sequencing is not only an important tool in genomic analysis but also in other applications, such as genetic identification, forensic analysis, genetic counseling and medical diagnostics. With respect to the area of medical diagnostics, disorders, susceptibilities to disorders and prognoses of disease conditions can be correlated with the presence or absence of particular DNA sequences (or the degree of variation in DNA sequences) at one or more genetic loci. Examples of such phenomena include human leukocyte antigen (HLA) sequence variation, cystic fibrosis, tumor progression and heterogeneity, p53 proto-oncogene mutations and ras proto-oncogene mutations. Recently, a sequence of the whole human genome of 3×109 bp was determined.
Various approaches to sequencing exist. Single molecule DNA analysis methods based on acquiring data from single molecules allow for the gathering of sequence information and generally fall into two categories: (a) the observation of interactions of DNA polymerase, RNA polymerase or exonucleases with DNA, and (b) the measurement of the physical properties of nucleotides in the DNA strand, typically based on ultra high resolution scanned probe microscopy or studies of DNA passing through nanopores (Li et al, Nat Mater 2:611-615 (2003)). Related methods have been described, which include monitoring the incorporation of fluorescently-labeled nucleotides into individual DNA strands by spFRET (Braslavsky et al, Proc Natl Acad Sci USA 100:3960-3964 (2003)) or monitoring of the fluorescently labeled nucleotides released from DNA by an exonuclease in a flow system, or nanopore-based analysis of DNA. Single-molecule DNA analysis can be performed in parallel on a solid support, and, in one implementation, it involves the incorporation of Cy3-labeled reversible chain-terminating dNTP with a cleavable disulfide bond. Sequence information could be derived from thousands of individual DNA molecules spread on a glass slide using a sensitive CCD camera.
Fluorescent dyes have been used in a variety of DNA sequencing techniques. A fluorophore moiety or dye is a molecule capable of generating a fluorescence signal. A quencher moiety is a molecule capable of absorbing the fluorescence energy of an excited fluorophore, thereby quenching the fluorescence signal that would otherwise be released from the excited fluorophore. In order for a quencher to quench an excited fluorophore, the quencher moiety must be within a minimum quenching distance of the excited fluorophore moiety at some time prior to the fluorophore emitting light.
The decreased fluorescence of a fluorophore moiety, a result of a collision or direct interaction with a quencher, is due mainly to a transfer of energy from the fluorophore in the excited state to the quencher. The extent of quenching depends on the concentration of quencher and is described by the Stem-Volmer relationship:Fo/F=1+Ksv[Q]wherein F0 and F correspond to the fluorescence in the absence and presence of a quencher, respectively, and [Q] is the quencher concentration. A plot of Fo/F versus [Q] yields a straight line with a slope corresponding to the Stem-Volmer constant, Ksv.
In general, fluorophore moieties have a high quantum yield and a large extinction coefficient so that the fluorescent dye can be used to measure small quantities of the component being detected. Fluorophore moieties preferably have a large Stokes shift (i.e., the difference between the wavelength at which the dye has maximum absorbance and the wavelength at which the dye has maximum emission) so that the fluorescent emission is readily distinguished from the light source used to excite the dye.
Fluorophore-quencher pairs have been incorporated into oligonucleotide probes in order to monitor biological events based on the fluorophore and quencher being separated or brought within a minimum quenching distance of each other. For example, probes have been developed wherein the intensity of the fluorescence increases due to the separation of the fluorophore-quencher pair. Probes have also been developed which lose their fluorescence because the quencher is brought into proximity with the fluorophore. These fluorophore-quencher pairs have been used to monitor hybridization assays and nucleic acid amplification reactions, especially polymerase chain reactions (PCR), by monitoring either the appearance or disappearance of the fluorescence signal generated by the fluorophore molecule.
A need exists for acquiring data related to sequences of nucleic acid by determining positions of certain codons along the polynucleotide chain. Therefore, a need exists for a fluorescent molecule which senses a nucleic acid sequence with high nucleotide specificity, without getting incorporated into the nucleic acid. These and further needs are met by the present invention. Benefits and advantages of the present invention include quick readout, use of very small quantities of reagents, capabilities for parallel readout, and low cost of the assay with a wide applicability to many laboratory procedures, diagnostics and drug discovery.